1. Field of the Invention
The present invention relates to an on-line system base sequencing apparatus for introducing nucleic acid fragment samples, which are pretreated by the Sanger method using fluorescent primers (primers obtained by chemically bonding fluorescent materials as markers), into a sample introducing part of a slab type gel of a gel electrophoresis apparatus in units of end bases. The samples are simultaneously electrophoresed and the fluorescence is detected during the electrophoresis by excitation using an optical detection system that scans in a direction perpendicular to the electrophoresis direction. The base sequencing is then performed with a data processing unit programmed to carry out a number of functions to prevent the effects of smiling.
2. Description of the Background Art
In an on-line system base sequencing apparatus using a slab type gel, nucleic acid fragments which are previously treated by the Sanger method are electrophoresed in different electrophoresis lanes in response to the types A (adenine), G (guanine), T (thymine) and C (cytosine) of the end bases thereof.
In general, a gel electrophoresis apparatus creates an undesirable so-called "smiling" effect, which is a phenomenon wherein the electrophoresis speeds vary with electrophoresis lanes. If the signals are successively read from the electrophoresis lanes for the end bases A, G, T and C and the samples are directly base-sequenced with out accounting for the "smiling" effect, the sequence can be inverted and result in misreading thereof.
It is believed that "smiling" is mainly caused by varying temperature distributions in the electrophoresis lanes caused by Joule heating, which is generated as the result of electrophoresis. In order to prevent "smiling", there has been proposed a method wherein a metal plate is placed in close contact with the electrophoresis plate thereby evening the temperature distribution. Another methods of solving varying temperature distributions in the electrophoresis lanes involves storing the electrophoresis plate in a closed container and supplying air controlled at a constant temperature thereby homogenizing the temperature, as disclosed in Japanese Patent Laying-Open Gazette No. 2-143145 (1990).
While the mobility difference between 500 bases and 501 bases is 0.2% (=1/500), for example, it is necessary to control any temperature irregularity to be not more than 0.1.degree. C. in order to suppress the mobility difference caused by "smiling" to be not more than 0.2% by temperature control. In practice, this type of temperature control is very difficult to maintain.
Another problem associated with conducting a base electrophoresis involves electrophoresis gel containing electrolytic ammonium persulfate as a catalyst, which tends to migrate toward the side of an external electrode buffer following electrophoresis. If the concentration of this electrolyte is varied with position, differences in ionic strength can occur between the different electrophoresis lanes resulting in "smiling." This type of "smiling" resulting from non-heterogeneous concentration of the electrolyte cannot be prevent by temperature control.
In addition to the aforementioned types of "smiling", misreading of the base sequence is also caused by nonheterogeneous sample introduction slots of the electrophoresis lanes. In general, electrophoresis distances (i.e. distances between sample introduction slots and a detection part) of an on-line system fluorescent DNA sequencing apparatus are about 200 to 500 mm. At the sample introduction slots, sample positions can easily be displaced by 1 to 2 mm from each other by differences in horizontal position of the gel formation, penetration of urea within the slots, and the like. A difference of 1 mm at the sample introduction slots corresponds to a difference of 0.2% (1/500) assuming the electrophoresis distance to be 500 mm. This is equal to the mobility difference between bases 500 and 501. It is impossible to prevent such mobility difference by temperature control.